We have assessed Tim-3 expression on different subsets of mononuclear cells, including CD4+ T cells, CD8+ T cells, NK cells (CD56+), B cells (CD19+), monocytes (CD14+), and myeloid DCs (mDCs; CD11c+) isolated from PBMCs of six melanoma patients and six healthy donors. We found that Tim-3 expression is up-regulated on the surface of NK cells, monocytes, and mDCs in melanoma patients and healthy donors. However, within these cell subsets, no significant difference was observed for Tim-3 expression between melanoma patients and healthy donors (Fig. S1).

Ex vivo blockade of the Tim-3–Tim-3L pathway enhances cytokine production by NY-ESO-1–specific CD8+ T cells. (A and B) Representative dot plots from one melanoma patient (A) and summary data for all melanoma patients (n = 8; B) showing the percentages of A2/NY-ESO-1 157–165 tet+ CD8+ T cells that produce IFN-γ and TNF among total NY-ESO-1–specific CD8+ T cells after short ex vivo stimulation with cognate peptide in the presence of blocking anti–Tim-3 and/or anti–PD-L1 mAbs or an isotype control antibody (IgG). (C) Summary data for melanoma patients (n = 6) showing the percentages of A2/CMV 495–503 tet+ CD8+ T cells that produce IFN-γ and TNF among total CMV-specific CD8+ T cells after short ex vivo stimulation with cognate peptide in the presence of blocking anti–Tim-3 and/or anti–PD-L1 mAbs or an isotype control antibody (IgG). The p-values were calculated using the Wilcoxon signed rank test. Data shown are representative of two independent experiments performed in duplicate.

We next assessed the impact of Tim-3 expression on cytokine production by NY-ESO-1–specific CD8+ T cells after a multiday in vitro stimulation (IVS) with peptide-pulsed APCs. PBMCs from nine melanoma patients with spontaneous NY-ESO-1–specific CD8+ T cell response were incubated for 6 d with NY-ESO-1 157–165 peptide in the presence of mAbs against Tim-3 and/or PD-L1 or IgG control antibodies. After 6 d, cells were shortly restimulated (6 h) with NY-ESO-1 peptide or HIV peptide as control before evaluating cytokine production by A2/NY-ESO-1 157–165 tet+ CD8+ T cells. We observed a significant increase in the frequencies of NY-ESO-1–specific CD8+ T cells that produced IFN-γ and TNF (P = 0.0039 and P = 0.0091, respectively) after incubation in the presence of cognate peptide and anti–Tim-3 mAbs, as compared with cognate peptide and IgG control (Fig. 4, A and B), resulting in a 1.6- and 1.8-fold change in the frequencies of IFN-γ– and TNF-producing NY-ESO-1–specific CD8+ T cells, respectively (Fig. 4 C). The frequency of IL-2–producing NY-ESO-1–specific CD8+ T cells increased in a majority of patients in the presence of cognate peptide and anti–Tim-3 mAbs alone, albeit the difference was not statistically significant when compared with IgG control (P = 0.1829). We further observed a significant increase in the frequencies of not only IFN-γ– and TNF- but also IL-2–producing NY-ESO-1–specific CD8+ T cells in the presence of both anti–Tim-3 and anti–PD-L1 mAbs when compared with IgG control antibodies (P = 0.0039), anti–Tim-3 mAbs alone (P = 0.0283, P = 0.0117, and P = 0.0418, respectively), and anti–PD-L1 mAbs alone (P = 0.0177, P = 0.0091, and P = 0.0128, respectively; Fig. 4, A and B and not depicted), suggesting a synergistic effect of Tim-3–Tim-3L and PD-1–PD-L1 blockades on NY-ESO-1–specific CD8+ T cell expansion. This increase resulted in a 1.9-, 2.3-, and 2.4-fold change in the frequencies of IFN-γ-, TNF-, and IL-2–producing NY-ESO-1–specific CD8+ T cells, respectively, when compared with IgG control antibody (Fig. 4 C). As control, PBMCs were incubated with an irrelevant peptide for 6 d in the presence of anti–Tim-3 or anti–PD-L1 mAbs. We did not observe a significant effect of Tim-3 or PD-1 pathway blockade on the frequency of cytokine-producing NY-ESO-1–specific CD8+ T cells after restimulation with cognate peptide (Fig. S3 B). Notably, we observed that Tim-3–Tim-3L blockade increased the percentages of NY-ESO-1–specific CD8+ T cells that produced TNF and IL-2, but not IFN-γ, among total NY-ESO-1–specific CD8+ T cells compared with incubation with IgG control antibody (P = 0.0090, P = 0.0128, and P = 0.1094, respectively; Fig. S4). This observation is in line with our data showing that Tim-3–Tim-3L blockade ex vivo enhances the functionality of NY-ESO-1–specific CD8+ T cells. Although PD-1–PD-L1 blockade alone did not significantly change the capacity of NY-ESO-1–specific CD8+ T cells to produce cytokines (i.e., no increase in the percentages of cytokine-producing cells among total NY-ESO-1–specific CD8+ T cells; Fig. S4), we observed that Tim-3–Tim-3L blockade in combination with PD-1–PD-L1 blockade augmented the percentages of NY-ESO-1–specific CD8+ T cells that produced TNF and IL-2 compared with incubation with IgG control antibody (P = 0.0078 and P = 0.0039, respectively) or Tim-3–Tim-3L blockade alone (P = 0.0433 and P = 0.0117, respectively; Fig. S4). This increase resulted in a 1.3- and 2.2-fold change in the percentages of TNF- and IL-2–producing NY-ESO-1–specific CD8+ T cells among total NY-ESO-1–specific CD8+ T cells, respectively, when compared with IgG control antibody (Fig. S4), indicating a synergistic effect of Tim-3–Tim-3L and PD-1–PD-L1 blockades. Notably, we observed a significant increase in the expression of PD-1 and Tim-3 by NY-ESO-1–specific CD8+ T cells after a 6-d IVS in the presence of cognate peptide and IgG control antibodies as compared with incubation with an irrelevant peptide (Fig. S5). These findings are in line with our previous observation of Tim-3 and PD-1 up-regulation by activated NY-ESO-1–specific CD8+ T cells. Interestingly, Tim-3 or PD-1 blockade in the presence of cognate peptide induced a modest but significant increase in PD-1 and Tim-3 expression, respectively, by NY-ESO-1–specific CD8+ T cells, but not by NY-ESO-1 tet− CD8+ T cells, compared with incubation with cognate peptide and IgG control antibodies (Fig. S5). The up-regulation of Tim-3 and PD-1 expression by NY-ESO-1–specific CD8+ T cells after PD-1 or Tim-3 blockade in the presence of cognate peptide is likely caused by higher levels of T cell activation and may possibly contribute to the synergistic effects of Tim-3 and PD-1 blockades.

Next, we evaluated whether Tim-3–Tim-3L blockade alone or in combination with PD-1–PD-L1 blockade increased proliferation of NY-ESO-1–specific CD8+ T cells in response to the cognate antigen in a multiday IVS. CFSE-labeled PBMCs from nine melanoma patients with spontaneous NY-ESO-1–specific CD8+ T cell response were stimulated for 6 d with NY-ESO-1 157–165 peptide in the presence of mAbs against Tim-3 and/or PD-L1 or IgG control antibodies. As control, PBMCs were incubated for 6 d with HIVpol 476–484 peptide. As shown in Fig. 5 A for two melanoma patients and in Fig. 5 (B and C) for nine patients, the addition of anti–Tim-3 mAbs augmented the frequencies of CFSElo and total A2/NY-ESO-1 157–165 tet+ CD8+ T cells stimulated in the presence of cognate peptide as compared with peptide and IgG control antibody (P = 0.0117 and P = 0.0039, respectively), resulting in a 1.9- and a 1.6-fold change in the frequencies of CFSElo and total A2/NY-ESO-1 157–165 tet+ CD8+ T cells, respectively (Fig. 5, D and E). In line with previous findings (Fourcade et al., 2009), PD-1–PD-L1 blockade in the presence of cognate peptide enhanced proliferating and total NY-ESO-1–specific CD8+ T cells (P = 0.0117 and P = 0.0039, respectively; Fig. 5 A and not depicted), resulting in a 1.7- and a 1.5-fold change in the frequencies of CFSElo and total A2/NY-ESO-1 157–165 tet+ CD8+ T cells, respectively (Fig. 5, D and E). Importantly, blockade of both Tim-3–Tim-3L and PD-1–PD-L1 pathways further increased the frequencies of CFSElo and total A2/NY-ESO-1 157–165 tet+ CD8+ T cells as compared with stimulation with IgG control antibodies (P = 0.0078 and P = 0.0039, respectively; Fig. 5, A–C), Tim-3 blockade alone (P = 0.0078 and P = 0.0039, respectively), or PD-L1 blockade alone (P = 0.0324 and P = 0.0078, respectively). This synergistic effect resulted in the highest increases in the frequencies of CFSElo and total A2/NY-ESO-1 157–165 tet+ CD8+ T cells (2.6- and 2.0-fold, respectively) as compared with stimulation with IgG control antibody (Fig. 5, D and E). No proliferation of NY-ESO-1 157–165-specific CD8+ T cells was observed after a 6-d IVS with an irrelevant peptide with or without Tim-3 and/or PD-1 blockade (Fig. S3 C).

Blockade of the Tim-3–Tim-3L pathway alone or in combination PD-1–PD-L1 blockade with prolonged antigen stimulation increases the frequency of proliferating and total NY-ESO-1–specific CD8+ T cells. Representative flow cytometry analysis from two melanoma patients showing percentages of CFSElo A2/NY-ESO-1 157–165 tet+ CD8+ T cells among total CD8+ T cells (A) and pooled data from melanoma patients (n = 9) showing the variation in the numbers of CFSElo (B) and total (C) A2/NY-ESO-1 157–165 tet+ cells for 106 CD8+ T cells. CFSE-labeled PBMCs were incubated for 6 d with NY-ESO-1 157–165 peptide or HIVpol 476–484 peptide and blocking anti–Tim-3 (aTim-3), and/or anti–PD-L1 (aPD-L1) mAbs or an isotype control antibody (IgG). (D and E) Fold change of the frequencies of CFSElo (D) and total (E) A2/NY-ESO-1 157–165 tet+ CD8+ T cells after 6-d IVS with cognate peptide and blocking anti–Tim-3 (aTim-3) and/or anti–PD-L1 (aPD-L1) mAbs (n = 9). The ratio of the percentages of CFSElo and total A2/NY-ESO-1 157–165 tet+ CD8+ T cells in the presence of indicated antibody treatment and isotype control antibody is shown. The p-values were calculated using the Wilcoxon signed rank test. Data shown are representative of two independent experiments performed in duplicate.

DISCUSSION

In this study, we show that Tim-3+PD-1+ NY-ESO-1–specific CD8+ T cells represent a highly dysfunctional population of tumor-induced T cells in patients with advanced melanoma. We first observed that Tim-3 expression is up-regulated on tumor-induced NY-ESO-1–specific CD8+ T cells and on Flu-specific CD8+ T cells in advanced stage melanoma patients as compared with CMV-specific and EBV-specific effector and effector/memory CD8+ T cells. Strikingly, and in contrast not only to total EBV- and CMV-specific CD8+ T cells but also to Flu-specific CD8+ T cells, the majority of Tim-3+ NY-ESO-1–specific CD8+ T cells up-regulates PD-1 expression. Therefore, unlike virus-specific CD8+ T cells evaluated in our study and HIV-specific CD8+ T cells (Jones et al., 2008), spontaneous Tim-3+ NY-ESO-1–specific CD8+ T cells coexpress PD-1 in patients with advanced melanoma.

One critical finding is that Tim-3+PD1+ NY-ESO-1–specific CD8+ T cells are more dysfunctional than Tim3−PD-1+ and Tim3−PD-1− CD8+ T cells, as they produced significantly less IFN-γ, TNF, and IL-2 ex vivo. We found no significant difference in terms of cytokine production between Tim3−PD-1− and Tim-3−PD-1+ NY-ESO-1–specific CD8+ T cells, suggesting that PD-1 up-regulation alone without Tim-3 up-regulation is not directly associated with T cell dysfunction (i.e., cytokine secretion). This observation is in line with our previous demonstration that PD-1 acts as a regulator of NY-ESO-1–specific CD8+ T cell expansion upon chronic antigen exposure and has no major impact on their functionality on a cell-per-cell basis (Fourcade et al., 2009). In one melanoma patient with very high levels of spontaneous NY-ESO-1–specific CD8+ T cells, we found that Tim-3+PD-1− NY-ESO-1–specific CD8+ T cells produced less cytokines than Tim-3−PD-1− NY-ESO-1–specific CD8+ T cells, suggesting that Tim-3 up-regulation alone by tumor antigen–specific CD8+ T cells defines a group of dysfunctional T cells independently of PD-1 up-regulation. Importantly, Tim-3+PD-1+ NY-ESO-1–specific CD8+ T cells produced significantly less cytokines than Tim-3+PD-1− NY-ESO-1–specific CD8+ T cells, supporting Tim-3+PD-1+ CD8+ T cells, as a more dysfunctional population than Tim-3+PD-1− CD8+ T cells. The low frequencies of Tim-3+PD-1− NY-ESO-1–specific CD8+ T cells did not allow us to extend this observation to the other melanoma patients included in our study. Interestingly, one study in mice with chronic persistent infections has shown that coexpression of multiple inhibitory receptors, including PD-1, LAG-3, 2B4, and CD160 by the same virus-specific CD8+ T cells was associated with lower T cell functions (Blackburn et al., 2009). Our findings further add to this observation and support that coexpression of Tim-3 and PD-1 is a marker of tumor-induced T cell dysfunction in patients with advanced melanoma.

We observed that blockade of the Tim-3–Tim-3L pathway ex vivo increased the percentages of NY-−ESO-1-specific CD8+ T cells that produced cytokines, supporting the role of the Tim-3 pathway in tumor antigen–specific T cell exhaustion/dysfunction. These findings are in line with one study demonstrating the role of Tim-3 blockade in improving Tim-3+ HIV-specific CD8+ T cell functions (Jones et al., 2008). Although we could not measure the percentages of cytokine-producing Tim-3+ NY-ESO-1–specific CD8+ T cells in wells containing the blocking anti–Tim-3 mAbs, it is tempting to speculate that the increased frequencies of cytokine-producing NY-ESO-1–specific CD8+ T cells occurred only within the Tim-3+ NY-ESO-1–specific CD8+ T cell compartment, which represents a fraction of total NY-ESO-1–specific CD8+ T cells (mean 28.8%) and includes a minority of cytokine-producing cells (mean 8.7 and 4.6% of IFN-γ– and TNF-producing Tim-3+ NY-ESO-1–specific CD8+ T cells among total NY-ESO-1–specific CD8+ T cells, respectively). Several lines of evidence support this assumption. First, the increase of cytokine-producing NY-ESO-1–specific CD8+ T cells was observed only in the presence of blocking anti–Tim-3 mAbs, but not blocking anti–PD-L1 mAbs, suggesting the critical role of the Tim-3–Tim3L pathway. Second, Tim-3 blockade in the presence of cognate peptide did not increase the frequency of cytokine-producing, CMV-specific CD8+ T cells, which express low levels of Tim-3, suggesting that the blockade’s effect requires Tim-3 up-regulation by antigen-specific T cells.

In addition, Tim-3 blockade in combination with prolonged antigen stimulation with cognate peptide increased the frequencies of cytokine-producing, proliferating, and total NY-ESO-1–specific CD8+ T cells, confirming the impact of the Tim-3–Tim-3L pathway on NY-ESO-1–specific T cell dysfunction. Strikingly, we also observed increased percentages of TNF- and IL-2–producing CD8+ T cells among total NY-ESO-1–specific CD8+ T cells, supporting the role of Tim-3 blockade in enhancing NY-ESO-1–specific CD8+ T cell functions on a cell-per-cell basis.

One novel finding is that Tim-3–Tim-3L blockade in combination with PD-1–PD-L1 blockade further increased the frequencies of not only IFN-γ– and TNF- but also IL-2–producing NY-ESO-1–specific CD8+ T cells, as well as the frequencies of proliferating and total NY-ESO-1–specific CD8+ T cells, upon prolonged stimulation with cognate antigen. It is likely that the enhanced capacity of NY-ESO-1–specific CD8+ T cells to produce IL-2 after Tim-3–Tim-3L blockade alone and, to a larger extent, after both Tim-3–Tim-3L and PD-1–PD-L1 blockades contributed to the increased frequencies of proliferating NY-ESO-1–specific CD8+ T cells. Collectively, our data demonstrate a synergistic effect of Tim-3–Tim-3L and PD-1–PD-L1 blockades on NY-ESO-1–specific CD8+ T cell functions.

In summary, our data demonstrate that Tim-3+PD-1+ NY-ESO-1–specific CD8+ T cells represent a highly dysfunctional population of tumor-induced T cells in patients with advanced melanoma. They show that Tim-3–Tim-3L blockade can partially restore NY-ESO-1–specific CD8+ T cell numbers and functions and acts in synergy with PD-1–PD-L1 blockade. Therefore, our data support the use of Tim-3–Tim-3L blockade in association with PD-1–PD-L1 blockade in immunotherapeutic interventions to reverse tumor-induced T cell exhaustion/dysfunction in patients with advanced melanoma. One caveat is the disruption of the Tim-3–Tim-3L pathway appears to contribute to autoimmunity (Koguchi et al., 2006). Therefore, it will be critical to carefully monitor the occurrence of serious autoimmune side effects in vivo.

MATERIALS AND METHODS

Study subjects.

Blood samples were obtained under the University of Pittsburgh Cancer Institute Internal Revue Board–approved protocols 00–079 and 05–140 from 19 HLA-A2+ patients with NY-ESO-1–expressing stage IV melanoma. NY-ESO-1 expression by patients’ tumors was assessed by RT-PCR and immunohistochemistry. All patients had serum NY-ESO-1–specific antibodies detected by ELISA assays. Frequencies of NY-ESO-1–specific CD8+ T cells were assessed ex vivo by flow cytometry using PE-labeled HLA-A2/NY-ESO-1 157–165 tetramers. Nine patients who exhibited spontaneous NY-ESO-1–specific CD8+ T cell responses were included in the study. The percentages of ex vivo detectable NY-ESO-1 157–165–specific CD8+ T cells isolated from patients’ PBMCs ranged from 0.012% to 2.5% of total CD8+ T cells (median 0.03%). PBMCs used in this study were obtained from patients with no prior immunotherapy.

Phenotypic analysis.

CD8+ T lymphocytes were purified from PBMCs of patients and healthy donors using MACS Column Technology (Miltenyi Biotec) and incubated with APC-labeled HLA-A2/NY-ESO-1 157–165, HLA-A2/CMV 495–503, HLA-A2/EBV-BMLF-1 280–288, or HLA-A2/Flu-M 58–66 tetramers. The purity of CD8+ T cells was always >95%. Tetramers were provided by the Ludwig Cancer Institute for Cancer Research, Lausanne branch. As a control for specificity of tetramer staining, we did not observe any positive staining of CD8+ T cells from patients when using an HLA-A2/HIVpol 476–484 tetramer (all patients were HIV sero-negative). The minimum percentage of antigen-specific CD8+ T cells detected ex vivo in patients using these tetramers was 0.010% of total CD8+ T cells. Next, cells were stained with PD-1-FITC or IgG1-FITC (BD), Tim-3-PE (R&D Systems), or IgG2a-PE (BD)–, CD3-ECD–, and CD8-PE-Cy7 (Beckman Coulter)–conjugated antibodies. A violet amine–reactive dye (Invitrogen) was used to assess the viability of the cells. Alternatively, after tetramer labeling, cells were stained with the following conjugated antibodies and reagents: PD-1-FITC, CD8-ECD or CD8-PE-Cy7, HLA-DR-ECD, CD45RO-ECD (Beckman Coulter), CD38-PerCp-Cy5.5, CCR7-biotin (BD), CD28-PerCp-Cy5.5, CD57-biotin (BD), CD45RA-PerCp-Cy5.5, CD27-Alexa750 (eBioscience), and streptavidin-Alexa Fluor 750 (Invitrogen). 2.5 million events were collected during flow cytometric analysis on a FACSAria machine (BD) and analyzed using FlowJo software (Tree Star, Inc.).

Statistics.

Statistical hypotheses were tested with the Wilcoxon signed rank test (for paired results from the same patient) using SAS v. 9.1. Tests were two-sided, and P ≤ 0.05 was considered significant. Because rank tests are not sensitive to the actual values in a comparison, only to their ranks, differing sets of values can produce identical p-values.

Online supplemental material.

Fig. S1 shows Tim-3 expression on different subsets of mononuclear cells isolated from PBMCs of six melanoma patients and six healthy donors. Fig. S2 depicts the activation and maturational status of Flu-M 58–66–specific CD8+ T cells isolated from PBMCs of melanoma patients. Fig. S3 shows that Tim-3 and PD-1 pathway blockades in the absence of cognate peptide stimulation have no effect on NY-ESO-1–specific CD8+ T cell cytokine production and proliferation. Fig. S4 demonstrates that blockade of the Tim-3/Tim-3L pathway alone or in combination with PD-1/PD-L1 blockade with prolonged antigen stimulation enhances NY-ESO-1–specific CD8+ T cell functionality. Fig. S5 depicts the effect of Tim-3 and PD-1 pathway blockades on PD-1 and Tim-3 expression by NY-ESO-1–specific CD8+ T cells. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20100637/DC1.

Acknowledgments

We thank Dr. Lisa Borghesi and Mr. Dewayne Falkner of the Flow Facility of the University of Pittsburgh, Department of Immunology for their technical support. We thank Drs. Ana Anderson, Lawrence P. Kane, and Daniel Olive for their critical reading of the manuscript. We also thank Ms. Lisa Spano for editorial assistance.

This work was supported by the National Institutes of Health/National Cancer Institute grants RO1CA90360 and RO1CA112198 (to H.M. Zarour) anda grant from teh Cancer Research Institute (to H.M. Zarour).

The authors have no conflicting financial interests.

Footnotes

Abbreviations used:

CGA

cancer-germline antigen

Flu

influenza

IVS

in vitro stimulation

mDC

myeloid DC

MFI

mean fluorescence intensity

PD-1

programmed death 1

PD-L1

programmed death ligand 1

Tim-3

T cell immunoglobulin and mucin-domain–containing molecule 3

Submitted: 31 March 2010

Accepted: 3 August 2010

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